Marcel A. Baluda

Senescence-associated genes in normal human oral keratinocytes

The current study was undertaken to identify senescence-associated (SA) genes in cultured normal human oral keratinocytes (NHOK). Primary NHOK were serially subcultured in vitro as dispersed cells in low (0.15 mM) Ca(2+) medium until senescence. The SA genes of NHOK were identified by comparing the expression levels of 3195 human genes between exponentially replicating and senescing cultures. Approximately 5% of the screened genes were upregulated in senescing NHOK by a factor greater than 3 compared with rapidly dividing NHOK culture. Among them, we identified discrete gene groups, i.e., cyclin-dependent kinase inhibitors, G-protein-coupled receptors, apolipoproteins, matrix metalloproteinases, and mitochondrial proteins. To validate the microarray results, we confirmed the enhanced expression of a few selected SA genes, i.e., gpr1, apo-D, apo-E, apo-L, mmp-1, mmp-3, cyb561, cyp1b1, and cyp4b1, by reverse transcription-PCR. These SA genes were upregulated in three independent cultures of NHOK at high population doubling (PD) levels compared with those of low PDs. The enhanced expression of these SA genes was also found in senescing NHOK maintained in 3T3 feeder cell system, as well as in the chemically defined medium containing low Ca(2+). These results indicate that the onset of senescence in NHOK is associated with altered expression of the SA genes, which represent discrete gene groups, independently of the donor variation or culture conditions.

Introduction of Human Telomerase Reverse Transcriptase to Normal Human Fibroblasts Enhances DNA Repair Capacity

Purpose: From numerous reports on proteins involved in DNA repair and telomere maintenance that physically associate with human telomerase reverse transcriptase (hTERT), we inferred that hTERT/telomerase might play a role in DNA repair. We investigated this possibility in normal human oral fibroblasts (NHOF) with and without ectopic expression of hTERT/telomerase. Experimental Design: To study the effect of hTERT/telomerase on DNA repair, we examined the mutation frequency rate, host cell reactivation rate, nucleotide excision repair capacity, and DNA end-joining activity of NHOF and NHOF capable of expressing hTERT/telomerase (NHOF-T). NHOF-T was obtained by transfecting NHOF with hTERT plasmid. Results: Compared with parental NHOF and NHOF transfected with empty vector (NHOF-EV), we found that (a) the N-methyl-N′-nitro-N-nitrosoguanidine-induced mutation frequency of an exogenous shuttle vector was reduced in NHOF-T, (b) the host cell reactivation rate of N-methyl-N′-nitro-N-nitrosoguanidine-damaged plasmids was significantly faster in NHOF-T; (c) the nucleotide excision repair of UV-damaged DNA in NHOF-T was faster, and (d) the DNA end-joining capacity in NHOF-T was enhanced. We also found that the above enhanced DNA repair activities in NHOF-T disappeared when the cells lost the capacity to express hTERT/telomerase. Conclusions: These results indicated that hTERT/telomerase enhances DNA repair activities in NHOF. We hypothesize that hTERT/telomerase accelerates DNA repair by recruiting DNA repair proteins to the damaged DNA sites.

Differential gene expression in neoplastic and human papillomavirus-immortalized oral keratinocytes.

We have previously demonstrated that normal human oral keratinocytes immortalized by transfection with human papillomavirus type-16 Dna became tumorigenic after exposure to a chemical carcinogen. In an effort to detect differentially regulated genes associated with this transition from the immortal to the malignant phenotype, we employed representational differences analysis (a PCR-coupled subtractive hybridization technique). After analysing 50 colonies, 12 putative messages were identified. Northern analysis comparison using the identified cDNAs as probes was made between normal human oral keratinocyte, papillomavirus-immortalized human oral keratinocytes (HOK-16B), a neoplastic cell line derived from HOK-16B (HOK-16B-BaP-T) and the human oral cancer cell lines Hep-2, SCC-9 and Tu-177. We found that mRNAs encoding for cyclophilin A, c-myc binding protein 1, the heat shock protein 90α and one unknown transcript were up-regulated in the oral cancer cell lines analysed as well as in HOK-16B cells. We also detected a downregulation of the mRNAs encoding the skin-derived antileukoproteinase SKALP/elafin, the translationally regulated p23 protein and one unknown transcript. Whether these messages are associated to the neoplastic conversion of human keratinocytes remains to be determined.

Senescence occurs with hTERT repression and limited telomere shortening in human oral keratinocytes cultured with feeder cells

We investigated the phenotypic and molecular alterations in normal human oral keratinocytes (NHOK) during in vitro replication in two different culture conditions. The cells were cultured either in chemically defined Keratinocyte Growth Medium (KGM) without feeder layers or in serum‐containing flavin‐adenine dinucleotide (FAD) medium with feeder layers. Primary NHOK underwent 22 ± 3 population doublings (PDs) in KGM and 42 ± 4 PDs in FAD medium, reflecting 52% increase in replication capacity with feeder layers. In both culture conditions, exponentially replicating NHOK demonstrated telomerase activity and expression of human telomerase reverse transcriptase (hTERT) gene. Telomerase activity and hTERT expression were rapidly diminished in senescing NHOK, which exhibited small decrease of telomere length for the remaining limited cellular replications until the complete arrest of cell division. However, telomere length in senescent NHOK was 6.7 ± 0.5 kilobase pairs (kbps), significantly longer than that (5.12 kbps) of senescent human fibroblasts. The onset of senescence was accompanied with marked induction of p16INK4A, and this occurred in both culture systems using either KGM or FAD medium. These results indicate that replicative senescence of NHOK is associated with loss of telomerase activity followed by limited telomere shortening.

The telomeric length and heterogeneity decrease with age in normal human oral keratinocytes.

We have examined the telomere length in NHOK explanted from 28 donors between the ages of 21 and 84 years. Genomic DNA was isolated from exponentially replicating NHOK and digested with HinFI to yield terminal restriction fragments (TRF). The TRF length ranged from 4.1 to 7.0 kbp with a mean of 5.3 +/- 0.8 kbp, which was significantly shorter than that (8.9 +/- 1.0 kbp) of normal human oral fibroblasts (NHOF). The TRF length was inversely correlated to the increase of donor age in NHOK (m=-23 bp per year; r=-0.60; P<0.001). Also, the heterogeneity of TRF length in cultured NHOK decreased with increased donor age (r=-0.38, P<0.05). These data indicated that clonogenic NHOK cells had replicated in situ and showed a progressive shortening of TRF length. The short telomere length and decreased telomeric length heterogeneity in immortalized cells suggested that there is a critical minimum for cell survival.

Enhanced activity of cloned hamster TERT gene promoter in transformed cells.

In 7,12-dimethylbenz[a]anthracene-treated hamster pouch epithelial cells, telomerase activity increased within 1 week of treatment and reached a 6-7-fold increase within 3 weeks. To investigate this phenomenon, we have cloned and sequenced the hamster telomerase catalytic subunit (hamTERT) promoter. Transient transfection with different genomic segments upstream of the ATG translation initiation codon linked to the luciferase reporter gene mapped the core promoter within a 250 bp region. Three major transcription initiation sites and several minor sites were found between -42 and -140 bp relative to the ATG site. Like the human and murine TERT promoters, the hamTERT promoter lacks TATA and CAT boxes and all three promoters share similar regulatory factor binding sites. DNase I footprint analysis revealed six protected regions which contain sequences homologous with known transcription factor binding sites. Three protein binding regions (I, II, and III) were essential for the promoter activity. Regions I and III bound to Sp1 and Sp3 transcriptional factors, whereas region II bound to an unknown factor. Transient transfection of a promoter-luciferase plasmid into Drosophila SL2 cells showed that Sp1 and Sp3 regulated the hamster TERT promoter in a concentration-dependent and synergistic manner. Telomerase activity showed a 2-4-fold and 8-10-fold increase in immortalized cells and tumor cells, respectively, but hamTERT expression was only increased 1.7-fold and 2.4-fold, respectively, in the same cells.

Telomere shortening does not occur during postmaturational aging in situ in normal human oral fibroblasts

We investigated whether the telomere length, i.e. mean terminal restriction fragment (TRF) length, decreases during in situ aging in normal human oral fibroblasts (NHOF). For this purpose, NHOF cultures were established from 50 different donors and tested after 14 population doublings (PD) when the cells were replicating exponentially. Telomere-specific Southern blotting and digital quantitation showed that the mean (+/-standard error (S.E.)) TRF length of all tested cultures was 7.72+/-0.17 kbps. The plot of TRF mean length versus donor age showed high variability in individual length with an apparent average decline of -7.8 bp per year of age, which was not statistically significant (r=0.11; P>0.1). These data indicate that telomere shortening does not occur during donor aging in situ, and therefore, is not physiologically relevant for NHOF.

The role of the bursa-dependent lymphoid tissue in oncogenesis by avian myeloblastosis virus

Abstract The bursa-dependent lymphoid system appears to be responsible for the development of resistance to tumor formation by avian myeloblastosis virus (AMV) in chickens. The pattern of development of resistance to tumor induction by AMV as a function of age has been determined. It parallels the development of the capacity to synthesize circulating antibodies. Hormonal bursectomy by injection of 19-nortestosterone (19-NT) into chick embryos prevents the normal development of resistance to this tumor virus. In addition, there is no difference in susceptibility to AMV between normal and 19-NT treated 1-day-old chick in which the bursa-dependent lymphoid tissue is not yet developed.

Expression of a truncated v-myb product in transformed chicken embryo fibroblasts

Transformed cells have been isolated after transfection of chicken embryo fibroblasts (CEF) with the DNA of a recombinant clone (KXA 3457) in which the v‐myb sequences are flanked by the two AMV‐LTRs. Abnormal myb‐specific RNA species and myb‐related polypeptides were found to be expressed in these cells, suggesting that transformation of CEF by v‐myb might require alterations of the oncogene product.

Homology between HL-23V and primate viruses and search for proviral DNA sequences of simian sarcoma associated virus and baboon endogenous virus in DNA from human leukemic cells

Abstract A type C virus isolate (HL-23V) from leukemic cells is a mixture of baboon endogenous virus (BAV) and simian sarcoma associated virus (SSAV). There is 13% or less homology between BAV 30S RNA and SSAV 30S RNA. RNA (30S) isolated from HL-23 virions appears to consist only of BAV 30S RNA and SSAV 30S RNA. DNA from the spleen or peripheral blood leukemic cells of 25 different leukemia patients, DNA from the spleen, kidney or lung of 15 different patients with other neoplasias and DNA from normal tissues of 14 different individuals were examined for the presence of SSAV or BAV proviral DNA sequences. Cellular DNA excess hybridization to purified 3 H-labeled 30S viral RNA revealed that SSAV-RNA or BAV-RNA hybridized to the same extent with leukemic human DNAs and normal human DNAs.